Reduced FK228 and use thereof

ABSTRACT

The present invention relates to reduced FK228 of the formula (I) 
                         
wherein R 1  and R 2  are the same or different and each is a hydrogen atom or a thiol-protecting group, or a salt thereof, and a histone deacetylase inhibitor containing this compound, an expression potentiator and a reactivation promoter of a transgene, and pharmaceutical agents containing them as active ingredients. The reduced FK228 or a salt thereof has a strong histone deacetylase inhibitory activity and this compound can be used for the prophylaxis or treatment of various diseases, in which histone deacetylation is involved, and for the gene therapy of such diseases.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a 371 of International Patent Application No.PCT/JP01/05954, filed on Jul. 9, 2001, and claims priority to JapanesePatent Application No. 2000-216584, filed on Jul. 17, 2000

TECHNICAL FIELD

The present invention relates to a compound having a histone deacetylaseinhibitory activity and use thereof.

BACKGROUND ART

Histone deacetylase is a metallodeacetylation enzyme having Zn in anactive center (M. S. Finnin et al., Nature 401, 188–193 (1999)). Thisenzyme is considered to change affinity of various acetylated histonesfor DNA. The direct biological phenomenon brought about thereby is astructural change of chromatin. The minimum unit of chromatin structureis a nucleosome wherein 146 bp DNA is wound 1.8 times counterclockwisearound a histone octamer (H2A, H2B, H3 and H4, each 2 molecules, corehistone). The core histone stabilizes nucleosome structure byinteraction of the positive charge of the N-terminal of each histoneprotein with DNA. The acetylation of histone is controlled by thebalance between acetylation reaction, in which histone acetyltransferaseis involved, and deacetylation, in which histone deacetylase isinvolved. The acetylation of histone occurs in a lysin residue of theN-terminal of histone protein that is evolutionally well preserved.Consequently, it is considered that the core histone protein loseselectric charge of N-terminal, the interaction with DNA is attenuatedand the nucleosomal structure becomes unstable. Therefore, deacetylationof histone is considered to bring about the opposite, or stabilizationof nucleosomal structure. However, there are much to be clarified withregard to the degree of change of chromatin structure due to acetylationand the relationship thereof with the secondarily derivedtranscriptional control and the like.

On the other hand, a compound represented by the formula

(hereinafter to be also referred to as FR901228 substance) has beenreported to derive a potent antitumor activity by selectively inhibitinghistone deacetylase. Moreover, this substance causes high histoneacetylation in treated cells, as a result of which it derivestranscriptional control activity for various genes, cell cycleinhibitory activity and apoptosis inhibitory activity (JP-B-7-64872, H.Nakajima et al., Exp. Cell Res. 241, 126–166 (1998)). While there haveheretofore been various reports on histone deacetylase inhibitorsderived from naturally occurring substances, the FR901228 substance is afirst pharmaceutical agent that has connected histone acetylation withbiological phenomena expressed thereby, and whose clinical utility hasbeen agreed on. The FR901228 substance has a disulfide bond in amolecule.

It is an object of the present invention to provide a compound having astronger histone deacetylase inhibitory activity and a histonedeacetylase inhibitor comprising the compound. Another object of thepresent invention is to provide use of the compound having a histonedeacetylase inhibitory activity as a pharmaceutical agent.

DISCLOSURE OF THE INVENTION

As a result of the intensive studies done by the present inventors in anattempt to achieve the above-mentioned objects, it has been found that,by reducing the disulfide bond of the FR901228 substance into a thiolform, a stronger histone deacetylase inhibitory activity can beafforded, and further that this compound is useful as a pharmaceuticalagent, which resulted in the completion of the present invention.Accordingly, the present invention provides the following.

-   (1) A compound represented by the formula (I)

wherein R¹ and R² are the same or different and each is a hydrogen atomor a thiol-protecting group, or a salt thereof.

-   (2) The compound of the above-mentioned (1), wherein R¹ and R² are    each a hydrogen atom, or a salt thereof.-   (3) The compound of the above-mentioned (2), which is represented by    the formula (II)

wherein R¹ and R² are each a hydrogen atom (hereinafter to be alsoreferred to as an FR135313 substance), or a salt thereof.

-   (4) A production method of a compound of any of the above-mentioned    (1)–(3) or a salt thereof, which comprises a step for cleaving a    disulfide bond in a compound represented by the formula (III)

(hereinafter to be also referred to as FK228).

-   (5) The production method of the above-mentioned (4), wherein the    compound of the formula (III) is represented by the formula (IV)

(hereinafter to be also referred to as an FR901228 substance).

-   (6) The production method of the above-mentioned (5), which    comprises a step for culturing a bacterial strain belonging to the    genus Chromobacterium, which is capable of producing a compound of    the formula (IV), in an aqueous nutrient medium under aerobic    conditions and recovering the compound, and a step for cleaving a    disulfide bond in the recovered compound of the formula (IV).-   (7) A histone deacetylase inhibitor comprising a compound of any of    the above-mentioned (1)–(3), or a salt thereof.-   (8) A pharmaceutical composition for the treatment or prophylaxis of    tumor, inflammatory disorders, diabetes, diabetic complication,    homozygous thalassemia, fibrosis, cirrhosis, acute promyelocytic    leukemia (APL), organ transplant rejection or autoimmune disease,    which comprises a compound of any of the above-mentioned (1)–(3), or    a salt thereof, as an active ingredient.-   (9) An expression potentiator or reactivation promoter of a    transgene, which comprises a compound of any of the above-mentioned    (1)–(3), or a salt thereof, as an active ingredient.-   (10) The expression potentiator or reactivation promoter of a    transgene of the above-mentioned (9), which is a pharmaceutical    agent.-   (11) The expression potentiator or reactivation promoter of a    transgene of the above-mentioned (10), wherein the pharmaceutical    agent is for gene therapy.-   (12) A method for the treatment or prophylaxis of tumor,    inflammatory disorders, diabetes, diabetic complication, homozygous    thalassemia, fibrosis, cirrhosis, acute promyelocytic leukemia    (APL), organ transplant rejection or autoimmune disease, which    comprises administering a pharmaceutically effective amount of a    compound of any of the above-mentioned (1)–(3), or a salt thereof,    to patients.-   (13) A method for potentiating expression of a transgene or for    promoting reactivation of a transgene, which comprises administering    a pharmaceutically effective amount of a compound of any of the    above-mentioned (1)–(3), or a salt thereof, to patients.-   (14) The method of the above-mentioned (13), wherein the    administration to patients is for gene therapy.-   (15) Use of a compound of any of the above-mentioned (1)–(3), or a    salt thereof, for the production of a pharmaceutical composition for    the treatment or prophylaxis of tumor, inflammatory disorders,    diabetes, diabetic complication, homozygous thalassemia, fibrosis,    cirrhosis, acute promyelocytic leukemia (APL), organ transplant    rejection or autoimmune disease.-   (16) Use of a compound of any of the above-mentioned (1)–(3), or a    salt thereof, for the production of an expression potentiator of a    transgene or a reactivation promoter of a transgene.-   (17) The use of the above-mentioned (16), wherein the expression    potentiator of a transgene or the reactivation promoter of a    transgene is for gene therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound represented by the followingformula(I)

wherein R¹ and R² are the same or different and each is a hydrogen atomor a thiol-protecting group, or a salt thereof. Preferably, both R¹ andR² are hydrogen atoms, more preferably an FR135313 substance representedby the following formula

The details of the above-mentioned definitions and their preferableembodiments are given in the following.

The term “lower” used in the present specification means 1 to 6 carbonatoms, unless otherwise indicated.

In the present invention, a suitable thiol-protecting group is thatgenerally used in this field, which is exemplified by, but not limitedto, the following:

-   those that form thioether to protect thiol group, such as benzyl    group optionally having substituents [the substituent is exemplified    by lower alkoxy (e.g., methoxy etc.), acyloxy (e.g., acetoxy etc.),    hydroxy, nitro and the like], picolyl, picolyl-N-oxide,    anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl,    acyloxymethyl (e.g., pivaloyloxymethyl, tertiary    butoxycarbonyloxymethyl etc.) and the like;-   those that form monothio, dithio or aminothioacetal to protect thiol    group, such as lower alkoxymethyl (e.g., methoxymethyl,    isobutoxymethyl etc.), tetrahydropyranyl, benzylthiomethyl,    phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl and    the like;-   those that form thioester to protect thiol group, such as tertiary    butoxycarbonyl (BOC), acetyl and its derivative, benzoyl and its    derivative and the like;-   those that form carbamine acid thioester to protect thiol group,    such as carbamoyl, phenylcarbamoyl, lower alkylcarbamoyl (e.g.,    methylcarbamoyl, ethylcarbamoyl etc.) and the like; and the like.    More specifically, each protecting group described in PROTECTIVE    GROUPS IN ORGANIC SYNTHESIS, Second Edition, T. W. Greene, P. G. M.    Wuts WILEY-INTERSCIENCE is preferably used.

The above-mentioned compound of the formula (I) may have stereoisomersuch as optical isomer or geometric isomer based on an asymmetric carbonatom and a double bond, all of which isomers and mixtures thereof arealso encompassed in the present invention. Moreover, the compound of theformula (I) can form a salt, which is also encompassed in the presentinvention. The salt is a biologically acceptable salt that is generallynon-toxic, and is exemplified by salts with base and acid additionsalts, inclusive of salts with inorganic base such as alkali metal salt(e.g., sodium salt, potassium salt and the like), alkaline earth metalsalt (e.g., calcium salt, magnesium salt and the like), ammonium salt,salts with organic base such as organic amine salt (e.g., triethylaminesalt, diisopropylethylamine salt, pyridine salt, picoline salt,ethanolamine salt, triethanolamine salt, dicyclohexylamine salt,N′,N′-dibenzylethylenediamine salt and the like), inorganic acidaddition salt (e.g., hydrochloride, hydrobromide, sulfate, phosphate andthe like), organic carboxylic acid or sulfonic acid addition salt (e.g.,formate, acetate, trifluoroacetate, maleate, tartrate, fumarate,methanesulfonate, benzenesulfonate, toluenesulfonate and the like), saltwith basic or acidic amino acid (e.g., arginine, aspartic acid, glutamicacid and the like), and the like. Further, solvate compounds (e.g.,inclusion compound such as hydrate and the like) thereof are alsoencompassed in the present invention.

In the compound of the formula (I) of the present invention, thedisulfide bond of the compound (FK228) represented by the formula (III)is cleaved, and can be referred to as reduced FK228, or FK228 thiol form(hereinafter the series of the compounds of the present invention arealso generally referred to as an FK228 thiol form).

The present invention also provides a production method of the FK228thiol form of the present invention. The production method of the FK228thiol form of the present invention characteristically includes a stepfor cleaving the disulfide bond of FK228. The cleavage of this bond canbe conducted by a method known in this field to the degree that does notadversely influence the histone deacetylase inhibitory activity of theobtained FK228 thiol form, or by a method modified as necessary.

More specifically, the cleavage of the disulfide bond is achieved usinga thiol compound generally used for a reduction treatment of a proteingenerally having a disulfide bond, such as mercaptoethanol, thioglycolacid, 2-mercaptoethylamine, benzenethiol, parathiocresol, dithiothreitoland the like. Preferably, mercaptoethanol and dithiothreitol are used.An excess thiol compound can be removed by dialysis, gel filtration andthe like. Other than thiol compound, electrolysis, sodiumtetrahydroborate, lithium aluminum hydride, sulfite and the like may beused.

The above-mentioned reduction treatment is conducted as appropriate by aknown process depending on the kind of reducing agent. For example, whenmercaptoethanol or dithiothreitol is used, this reagent is added toFK228 and reacted at room temperature—under heating for 15min—overnight, preferably at room temperature overnight (seeBio-chemical Experiment Method 8, chemical modification of SH group,Masatsune Ishiguro, Japan Scientific Societies Press, IV, chemicalmodification of disulfide bond; Bio-chemical Experiment Method 10,quantitative determination of SH group, Hiroshi Matsumoto, ToyoKuninori, Japan Scientific Societies Press, III, reduction of SS bond,and the like).

A compound to be the starting material of FK 228 thiol form of thepresent invention, namely, FK228 or a salt thereof, is a known substanceand available. For example, FR901228 substance, which is one of thestereoisomers of FK228, can be obtained by culturing a bacterial strainbelonging to the genus Chromobacterium, which is capable of productionthereof, under aerobic conditions and recovering the substance fromculture broth. The bacterial strain belonging to the genusChromobacterium, which is capable of producing FR901228 substance, is,for example, Chromobacterium violaceum WB968 strain (FERM BP-1968). TheFR901228 substance can be obtained from this production strain accordingto JP-B-7-64872. The FR901228 substance is preferably recovered from abacterial strain belonging to the genus Chromobacterium, which iscapable of producing FR901228 substance, because it can be obtained moreeasily. In addition, a synthesized or semi-synthesized FR901228substance is also advantageous because further purification step isunnecessary or less. Alternatively, FK228 can be semi-synthesized orcompletely synthesized according to a method conventionally known. Morespecifically, the method reported by Khan W. Li, et al. (J. Am. Chem.Soc., vol. 118, 7237–7238 (1996)) can be used.

Another aspect of the FK228 thiol form of the present invention is acompound wherein R¹ and/or R² are/is a thiol-protecting group. Thiscompound can be prepared by introducing a thiol-protecting group intothe compound of the present invention wherein R¹ and R² are hydrogenatoms.

A suitable agent for introducing thiol-protecting group to be used inthis reaction is appropriately determined depending on the protectinggroup to be introduced. For example, those generally used, such aschloride of the corresponding protecting group (e.g., benzyl chloride,methoxybenzyl chloride, acetoxybenzyl chloride, nitrobenzyl chloride,picolyl chloride, picolyl chloride-N-oxide, anthryl methyl chloride,isobutoxymethyl chloride, phenylthiomethyl chloride etc.) and alcoholsof the corresponding protecting group (diphenylmethyl alcohol,adamanthyl alcohol, acetamidemethyl alcohol, benzamidomethyl alcoholetc.), dinitrophenyl, isobutylene, dimethoxymethane, dihydropyran,t-butyl chloroformate and the like can be mentioned.

This reaction can be carried out according to a conventionally knownmethod for a protecting group-introducing agent or a suitablecombination of such methods. The deprotection of the protecting group isalso known to those of ordinary skill in the art (PROTECTIVE GROUPS INORGANIC SYNTHESIS, Second Edition, T. W. Greene, P. G. M. WutsWILEY-INTERSCIENCE). As one specific example, when the introducing agentis benzyl chloride, protection of the thiol group is achieved byreaction in the presence of 2N sodium hydroxide and ethanol at 25° C.for 30 min, and deprotection thereof is achieved by treatment in thepresence of sodium and ammonia for 10 min.

FK228 thiol form of the present invention has a potent histonedeacetylase inhibitory activity, and is useful as a histone deacetylaseinhibitor in various mammals inclusive of human, such as monkey, mouse,rat, rabbit, swine, dog, horse, cow and the like.

Moreover, because of its histone deacetylase inhibitory activity, apharmaceutical composition containing the FK228 thiol form of thepresent invention is useful as an agent for the treatment or prophylaxisof the diseases (e.g., inflammatory disorder, diabetes, diabeticcomplication, homozygous thalassemia, fibrosis, cirrhosis, acutepromyelocytic leukemia (APL), protozoiasis and the like) induced byabnormal gene expression. In addition, the pharmaceutical composition ofthe present invention is useful as an antitumor agent andimmunosuppressant which prevents rejection of organ transplant andautoimmune diseases exemplified below. To be specific, the followingdiseases are targeted.

Rejection reactions by transplantation of organs or tissues such as theheart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, smallintestine, limb, muscle, nerve, intervertebral disc, trachea, myoblast,cartilage, etc.;

-   graft-versus-host reactions following bone marrow transplantation;-   autoimmune diseases such as rheumatoid arthritis, systemic lupus    erythematosus, Hashimoto's thyroiditis, multiple sclerosis,    myasthenia gravis, type I diabetes, etc.;-   infections caused by pathogenic microorganisms (e.g., Aspergillus    fumigatus, Fusarium oxysporum, Trichophyton asteroides, etc.);-   inflammatory or hyperproliferative skin diseases or cutaneous    manifestations of immunologically-mediated diseases (e.g.,    psoriasis, atopic dermatitis, contact dermatitis, eczematoid    dermatitis, seborrheic dermatitis, lichen planus, pemphigus, bullous    pemphigoid, epidermolysis bullosa, urticaria, angioedema,    vasculitides, erythema, dermal eosinophilia, lupus erythematosus,    acne, and alopecia areata);-   autoimmune diseases of the eye (e.g., keratoconjunctivitis, vernal    conjunctivitis, uveitis associated with Behcet's disease, keratitis,    herpetic keratitis, conical keratitis, corneal epithelial dystrophy,    keratoleukoma, ocular premphigus, Mooren's ulcer, scleritis, Graves'    ophthalmopathy, Vogt-Koyanagi-Harada syndrome, keratoconjunctivitis    sicca (dry eye), phlyctenule, iridocyclitis, sarcoidosis, endocrine    ophthalmopathy, etc.);-   reversible obstructive airways diseases [asthma (e.g., bronchial    asthma, allergic asthma, intrinsic asthma, extrinsic asthma, and    dust asthma), particularly chronic or inveterate asthma (e.g., late    asthma and airway hyper-responsiveness), bronchitis, etc.];-   mucosal or vascular inflammations (e.g., gastric ulcer, ischemic or    thrombotic vascular injury, ischemic bowel diseases, enteritis,    necrotizing enterocolitis, intestinal damages associated with    thermal burns, leukotriene B4-mediated diseases);-   intestinal inflammations/allergies (e.g., coeliac diseases,    proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's    disease and ulcerative colitis);-   food-related allergic diseases with symptomatic manifestation remote    from the gastrointestinal tract (e.g., migrain, rhinitis and    eczema);-   renal diseases (e.g., intestitial nephritis, Goodpasture's syndrome,    hemolytic uremic syndrome, and diabetic nephropathy);-   nervous diseases (e.g., multiple myositis, Guillain-Barre syndrome,    Meniere's disease, multiple neuritis, solitary neuritis, cerebral    infarction, Alzheimer's disease, Parkinson's disease, amyotrophic    lateral sclerosis (ALS), and radiculopathy);-   cerebral ischemic diseases (e.g., head injury, hemorrhage in brain    (e.g., subarachnoid hemorrhage, intracerebral hemorrhage), cerebral    thrombosis, cerebral embolism, cardiac arrest, stroke, transient    ischemic attack (TIA), and hypertensive encephalopathy);-   endocrine diseases (e.g., hyperthyroidism, and Basedow's disease);-   hematic diseases (e.g., pure red cell aplasia, aplastic anemia,    hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune    hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic    anemia, and anerythroplasia);-   bone diseases (e.g., osteoporosis);-   respiratory diseases (e.g., sarcoidosis, pulmonary fibrosis, and    idiopathic interstitial pneumonia);-   skin diseases (e.g., dermatomyositis, leukoderma vulgaris,    ichthyosis vulgaris, photosensitivity, and cutaneous T-cell    lymphoma);-   circulatory diseases (e.g., arteriosclerosis, atherosclerosis,    aortitis syndrome, polyarteritis nodosa, and myocardosis);-   collagen diseases (e.g., scleroderma, Wegener's granuloma, and    Sjögren's syndrome);-   adiposis;-   eosinophilic fasciitis;-   periodontal diseases (e.g., damage to gingiva, periodontium,    alveolar bone or substantia ossea dentis);-   nephrotic syndrome (e.g., glomerulonephritis);-   male pattern alopecia, alopecia senile;-   muscular dystrophy;-   pyoderma and Sezary syndrome;-   chromosome abnormality-associated diseases (e.g., Down's syndrome);-   Addison's disease;-   active oxygen-mediated diseases [e.g., organ injury (e.g., ischemic    circulation disorders of organs (e.g., heart, liver, kidney,    digestive tract, etc.) associated with preservation,    transplantation, or ischemic diseases (e.g., thrombosis, cardial    infarction, etc.));-   intestinal diseases (e.g., endotoxin shock, pseudomembranous    colitis, and drug- or radiation-induced colitis);-   renal diseases (e.g., ischemic acute renal insufficiency, chronic    renal failure);-   pulmonary diseases (e.g., toxicosis caused by pulmonary oxygen or    drugs (e.g., paracort, bleomycin, etc.), lung cancer, and pulmonary    emphysema);-   ocular diseases (e.g., cataracta, iron-storage disease (siderosis    bulbi), retinitis, pigmentosa, senile plaques, vitreous scarring,    corneal alkali burn);-   dermatitis (e.g., erythema multiforme, linear immunoglobulin A    bullous dermatitis, cement dermatitis);-   and other diseases (e.g., gingivitis, periodontitis, sepsis,    pancreatitis, and diseases caused by environmental pollution (e.g.,    air pollution), aging, carcinogen, metastasis of carcinoma, and    hypobaropathy)];-   diseases caused by histamine release or leukotriene C4 release;-   restenosis of coronary artery following angioplasty and prevention    of postsurgical adhesions;-   Autoimmune diseases and inflammatory conditions (e.g., primary    mucosal edema, autoimmune atrophic gastritis, premature menopause,    male sterility, juvenile diabetes mellitus, pemphigus vulgaris,    pemphigoid, sympathetic ophthalmitis, lens-induced uveitis,    idiopathic leukopenia, active chronic hepatitis, idiopathic    cirrhosis, discoid lupus erythematosus, autoimmune orchitis,    arthritis (e.g., arthritis deformans), or polychondritis);-   Human Immunodeficiency Virus (HIV) infection, AIDS;-   allergic conjunctivitis;-   hypertrophic cicatrix and keloid due to trauma, burn, or surgery.

Moreover, the FK228 thiol form of the present invention is useful as anexpression potentiator or reactivation promoter of a transgene due toits histone deacetylase inhibitory activity.

In the present invention, potentiation of the expression of a transgenemeans potentiation in the host cell of the expression of an exogenousgene transduced by genetic engineering into the cells of human andvarious animals (e.g., mouse, rat, swine, dog, horse, cow and the like).The potentiation of the expression of the transgene may be at the celllevel (i.e., in vitro) or at an individual level (i.e., in vivo), withpreference given to that in vivo.

As used herein, in vivo and in vitro means as these terms are used inthis field. That is, “in vivo” means that the target biologicalfunctions and responses are expressed within tissues of the living body,and “in vitro” means that such functions and responses are expressed ina test tube (tissue culture system, cell culture system, non-cell systemand the like).

In the present invention, reactivation of a transgene means release ofthe suppression of the expression (silencing) of an exogenous genetransduced by genetic engineering into the cells of human and variousanimals (e.g., mouse, rat, swine, dog, horse, cow and the like), and thepresent invention can promote the reactivation. Besides the release ofsilencing, the present invention can promote transcription activity of atransgene that shows stable expression at a constant level, andpotentiate the expression. Such effect is also encompassed in the“reactivation of the transgene” of the present invention. The promotionof the reactivation of the transgene may be at a cell level (i.e., invitro) or at an individual level (i.e., in vivo), with preference givento that in vivo.

An exogenous gene can be transduced by a method known in the pertinentfield. For example, transfer of DNA by physical method (microinjectionmethod, electroporation method and the like), transfer of DNA bychemical method (calcium phosphate method, DEAE-dextran method etc.),biological method (virus vector such as retrovirus and adenovirus, andthe like), new methods such as HVJ-liposome method and the like can bebeneficially used.

When the FK228 thiol form of the present invention or a salt thereof isused as a pharmaceutical agent, it can be used as a solid, semi-solid orliquid pharmaceutical preparation containing FK228 thiol form or a saltthereof as an active ingredient in admixture with an organic orinorganic carrier or excipient suitable for oral or parenteralapplication. The active ingredient can be admixed with a typical,non-toxic pharmaceutically acceptable carrier suitable for the dosageform, such as powder, tablet, pellet, capsule, suppository, liquid,emulsion, suspension, aerosol, spray and other form for use. Wherenecessary, auxiliary agent, stabilizer, tackifier and the like may beused. These carriers and excipients may be sterilized where necessary,or a sterilization treatment may be applied after formulation into apreparation. FK228 thiol form or a salt thereof are contained in theexpression potentiator or reactivation promoter in an amount sufficientto produce a desired effect on the condition that requires potentiationof the expression of a transgene or reactivation thereof. In particular,when the inventive expression potentiator and reactivation promoter of atransgene is used for a gene therapy, parenteral administration ispreferable, namely, intravenous administration, intramuscularadministration, direct administration into the tissue, intra-nostrilcavity administration, intradermal administration, administration intocerebrospinal fluid, administration into biliary tract, intravaginaladministration and the like. In addition, a liposome method capable ofdirect administration to the site and organ where-expression andreactivation of a transgene are requested, and the like can bepreferably used.

The therapeutically effective amount of the active ingredient FK228thiol form and a salt thereof varies and is determined depending on theage and condition of individual patient to be treated, and when it isused as an expression potentiator or a reactivation promoter of atransgene, on the kind of the transgene, and the kind of a disease wherepotentiation of the expression and promotion of reactivation of atransgene are requested.

The administration method of a pharmaceutical agent containing the FK228thiol form of the present invention or a salt thereof as an activeingredient is free of any particular limitation as long as it canprovide the desired effect, and, for example, the agent can beadministered orally or parenterally once a day or several times a day.When it is used for a gene therapy, the administration route mostsuitable for the expression and reactivation of the transgene isappropriately selected in consideration of the specific nature of use.For example, when it is used for a gene therapy of tumor, directadministration to the tumor cell (e.g., liposome method) is preferable.

The expression potentiator and reactivation promoter of a transgene ofthe present invention is characterized by the potentiation of theexpression of a transgene, as well as release of the suppression of thetransgene expression, wherein the interaction with the transgene is animportant factor for the exertion of the effect. Therefore, the timingof the administration of the transgene and the administration (in vivo,in vitro) to the subject of the expression potentiator or reactivationpromoter of the present invention are appropriately determined accordingto the desired effect. When the potentiation of the expression of atransgene is aimed, for example, the inventive transgene expressionpotentiator is preferably administered along with or after theadministration of the transgene. When the promotion of the reactivationof a gene already transduced is aimed, the inventive transgenereactivation promoter is preferably administered when the reactivationis needed after the administration of the transgene. When the expressionpotentiator or reactivation promoter of a transgene of the presentinvention is to be administered after the administration of thetransgene, the timing of the administration is appropriately determinedaccording to the desired effect and its level, and state of expressionof the gene previously transduced (level of expression, position of thetransgene and the like).

In particular, the expression potentiator and reactivation promoter of atransgene of the present invention can be beneficially applied to a genetherapy. For the gene therapy of cancer, for example, transfer of asuicide gene, DNA vaccine and the like can be applied. As the transferof a suicide gene, there is exemplified transfer of cytosine deaminase(enzyme to convert an anticancer agent, 5-fluorocytosine (5-FC) from aninactive type to an active type compound) gene into cancer cells. Theexpression of this gene in a cancer cell can be potentiated by thepresent invention (induction of anti-tumor effect by cancercell-specific and efficient conversion of 5-FC to an active type 5-FC).As the DNA vaccine, there is exemplified a tumor-associated antigen genespecifically expressed in a cancer cell. Transfer of the gene to acancer patient, or reactivation of an endogenous tumor-associatedantigen gene, expression of which is suppressed, or both of them,provide potentiation of the expression of the function of thetumor-associated antigen gene, which in turn enhances the immunity tothe cancer of the patient.

In a gene therapy of cancer, p53 gene, cytokine gene (e.g., IL2, IL12gene), antisense gene (K-ras antisense) and the like are also used. Forthe gene therapy of cystic fibrosis, CFTR gene can be used and for thegene therapy of hemophilia, a coagulant factor gene can be used.

EXAMPLES

The present invention is explained in more detail in the following byway of Examples. It is needless to say that the present invention is notlimited by these examples.

Production Example Production of FR135313

FR901228 isolated and purified according to the description ofJP-B-7-64872 was used as a starting substance. To a mixture of FR901228(51.6 mg, 95 μmol), water (40 ml) and acetonitrile (10 ml), was addeddithiothreitol (412 mg, 2.66 mmol), and the mixture was left standingovernight at room temperature. Acetonitrile was distilled away and themixture was purified by preparative HPLC (washing was conducted usingaqueous solution of 20% acetonitrile/0.05% trifluoroacetic acid andelution was conducted using an aqueous solution of 50%acetonitrile/0.05% trifluoroacetic acid).

The fractions containing the objective compound were recovered andlyophilized to give FR135313 as a powder (14.8 mg, yield 28.7%)

¹H-NMR(500 MHz, DMF-d₇) δ:9.35 (1H, br s, exchangeable), 8.15 (1H, br d,J=9 Hz, exchangeable), 8.01 (1H, br d, J=7 Hz, exchangeable), 6.83 (1H,d, J=7 Hz, exchangeable), 6.81 (1H, q, J=7 Hz), 5.72 (1H, m), 5.61–5.54(2H, m), 4.60(1H, dd, J=10 Hz, 5 Hz), 4.55 (1H, m), 4.15 (1H, dd, J=9Hz, 8 Hz), 2.97−2.88 (2H, m), 2.73–2.63 (2H, m), 2.55 (2H, m), 2.44 (1H,t, J=8 Hz, exchangeable), 2.34–2.27 (3H, m), 2.20 (1H, m), 2.08 (1H, t,J=8 Hz, exchangeable), 1.72 (3H, d, J=7 Hz), 0.98 (3H, d, J=7 Hz), 0.95(3H, d, J=7 Hz), 0.88 (3H, d, J=7 Hz), 0.87 (3H, d, J=7 Hz) MS m/e 654(M+TFA)

The purity of the objective compound was confirmed by

-   HPLC under the following conditions.    HPLC Conditions-   column: YMC-PACK ProC18 (YMC Co., Ltd), 4.6×150 mm-   elution: aqueous solution of 50% acetonitrile/0.05%-   trifluoroacetic acid-   flow rate: 1 ml/min-   detection : 214 nm, 254 nm-   retention time: 4.01 min (retention time of starting substance    FR901228 substance is 4.27 min)

Experimental Example Assay of Histone Deacetylase Activity

The histone deacetylase inhibitory activity of the FR135313 substancesynthesized in Production Example 1 was examined.

1. Test material·Test method

(1) Cell

Mouse breast cancer FM3A was supplied by Dr. Dai Ayusawa of MedicalDepartment, Yokohama City University. This cell was subcultured in an ESmedium containing 2% FBS (Flow Laboratories, hereinafter to be referredto as ES medium) at 37° C., in 5% CO₂.

(2) Pharmaceutical Agent

Sodium butyrate was purchased from Waco Pure Chemical Industries, Ltd.and [³H] sodium acetate was purchased from Amersham.

(3) Buffer and the Like

-   Lysis buffer (pH 6.5): 10 mM Tris-HCl (Sigma), 50 mM sodium    bisulfite (Nakarai Chemical, Ltd), 1% Triton X-100 (Nakarai    Chemical, Ltd), 10 mM magnesium chloride (Nakarai Chemical, Ltd),    8.6% sucrose (Nakarai Chemical, Ltd)-   Washing buffer (pH 7.4): 10 mM Tris-HCl, 13 mM EDTA (Sigma)-   HDA buffer (pH 7.5): 15 mM potassium phosphate (Nakarai Chemical,    Ltd), 5% glycerol, 0.2 mM EDTA    (4) Preparation of [³H] Acetylated Histone

The [³H] acetylated histone to be the substrate of histone deacetylasewas prepared by culturing 1×10⁸ cells of FM3A cell (suspended in 50 mlof ES medium) in the presence of 0.5 mCi/ml [³H] sodium acetate and 5 mMsodium butyrate at 37° C. in 5% CO₂ for 30 min, and immediatelyextracting histone fraction from the treated cells according to thefollowing method. The specific radioactivity was 0.45 μCi/mg histone.

(5) Extraction of Histone Protein from Cell

Extraction of histone protein from culture cell was conducted accordingto the method of Yoshida et al. (M. Yoshida et al., J. Biol. Chem. 265,17174–17179 (1990)). 1×10⁸ cells of FM3A cells labeled with [³H] sodiumacetate were recovered and washed once with PBS. The washed cells weresuspended in 1 ml of ice-cooled lysis buffer and ruptured by Douncehomoegnizer. The nucleus was collected by centrifugation at 1000 rpm for10 min, and washed 3 times with the lysis buffer and then once with thewashing buffer. The residue was suspended in 0.1 ml of ice-cooleddistilled water and concentrated sulfuric acid (Waco Pure ChemicalIndustries, Ltd.) was added to the final concentration of 0.4 N, and themixture was stood at 4° C. for 1 hr. The suspension was centrifuged in amicrocentrifugal machine at 15,000 rpm for 5 min, the supernatant wasrecovered, to which 1 ml of acetone was added, and the supernatant wasleft standing overnight at −20° C. The precipitate was recovered bycentrifugation in a microcentrifugal machine at 15,000 rpm for 10 min,and dried.

(6) Extraction of Crude Histone Deacetylase from Cell

Mouse histone deacetylase was pre-purified from FM3A cells. Suspendedcultured FM3A cells (concentration of 1×10⁶ cells/ml in ES medium 4L) inan 8L spinner flask were recovered by centrifugation and suspended in 40ml of HDA buffer. The cells were ruptured by a Dounce homoegnizer, andcell nucleus was recovered by centrifugation at 35,000×g for 10 min andfurther ruptured in 20 ml of 1 M ammonium sulfate solution. A cloudyrupture suspension was ultrasonicated and centrifuged to give atransparent extract, to which ammonium sulfate was added and theammonium sulfate concentration was raised to 3.5 M, whereby histonedeacetylase precipitated. The precipitate was dissolved in 10 ml of HDAbuffer, and dialyzed against 4 L of the same buffer. The dialysate wasbuffered with HDA buffer. It was applied to DEAE-cellulose (DE52, 25×85mm, Whatman) and eluted with 300 ml of NaCl by linear gradient (0–0.6M). The histone deacetylase activity was eluted as a single peakactivity in 0.2–0.3 M NaCl elution fraction. As a result, histonedeacetylase was purified to about 60 times specific activity.

(7) in Vitro Histone Acetylation Reaction

4 μl of [³H] acetylated histone (2500 cpm/5 μg) and 96 μl of crudehistone deacetylase fraction were admixed. An ethanol solution (1 μl) ofFR135313 substance prepared according to the above-mentioned ProductionExample was added to the mixture at various final concentrations, andthe mixture was reacted at 37° C. for 10 min. The reaction wasterminated by the addition of 10 μl of concentrated hydrochloric acid,and released [³H] acetic acid was extracted with 1 ml of ethyl acetate,from which 0.9 ml was added to 5 ml of toluene scintillation solutionand the radioactivity was measured.

(8) Result

The FR135313 substance, which is in a reduced form (thiol form), showedhistone deacetylase inhibitory activity as shown in IC₅₀ value of notmore than 1 ng/ml.

The thiol group showed a strong chelating action and therefore, theFR901228 substance that became a thiol form under the reductionenvironment, is considered to inhibit the activity of histonedeacetylase by its directivity to this enzyme, which is a metalloenzyme.

INDUSTRIAL APPLICABILITY

The compound of the formula (I), which is a reduced form (thiol form) ofFK228, particularly FR135313 substance, which is a reduced form (thiolform) of FR901228 substance, and salts thereof have a strong histonedeacetylase inhibitory activity, and are useful as a histone deacetylaseinhibitor or an agent for the prophylaxis or treatment of inflammatorydisorder, diabetes, diabetic complication, homozygous thalassemia,fibrosis, cirrhosis, acute promyelocytic leukemia (APL), organtransplant rejection or autoimmune disease, and further as an expressionpotentiator or reactivation promoter of a transgene.

By controlling the activity of thiol group, the histone deacetylaseinhibitory activity can be controlled, thereby enabling development of apharmaceutical agent suitable for various clinical applications.

1. A compound represented by the formula (I)

wherein R¹ and R² are the same or different and each is a hydrogen atomor a thiol-protecting group, or a salt thereof.
 2. The compound of claim1, wherein R¹ and R² are each a hydrogen atom, or a salt thereof.
 3. Thecompound of claim 2, which is represented by the formula (II)

wherein R¹ and R² are each a hydrogen atom, or a salt thereof.
 4. Amethod of producing a compound of claim 1 or a salt thereof, whichcomprises cleaving a disulfide bond in a compound represented by theformula (III)


5. The method of claim 4, wherein the compound of the formula (III) isrepresented by the formula (IV)


6. A method for the treatment of tumor, inflammatory disorders,diabetes, diabetic complication, homozygous thalassemia, fibrosis,cirrhosis, acute promyelocytic leukemia (APL), organ transplantrejection or autoimmune disease, which comprises administering apharmaceutically effective amount of a compound of claim 1, or a saltthereof, to a patient in need thereof.